In the last decade, major scientific advances in bioimaging based on the use of ultrashort (picosecond- and femtosecond-duration) light pulses have occurred, and initial demonstrations of short-pulse diagnosis and treatment of disease have also been reported. Examples include multiphoton microscopy, optical coherence tomography, and coherent anti-Stokes Raman spectroscopy (CARS) microscopy. A relative weakness of optical imaging techniques is their poor depth-penetration: imaging at visible wavelengths is limited to samples less than approximately one millimeter thick, and superficial tissue. Images of structures much deeper in tissue could be obtained with infrared wavelengths between 1000 and 1300 nm. Sources of ultrashort light pulses in this wavelength range are complicated and expensive, and this hinders the applications of nonlinear-imaging techniques. Fiber lasers that generate short pulses ideal for nonlinear microscopies will be developed. The goal of this project is to demonstrate femtosecond and picosecond fiber lasers that match or exceed the performance of the solid-state lasers currently employed in nonlinear microscopies, but with the major advantages of fiber: the sources will be compact, stable, user-friendly, compatible with endoscopic instruments, and inexpensive. The lasers will be developed and used in collaboration with experts in biomedical imaging at Cornell, Harvard, and Michigan State universities. Third-harmonic generation microscopy will be applied to in vivo characterization of morphological changes in cells caused by small-scale strokes in the rat cortex, while multiplex CARS microscopy will be employed to determine blood oxygenation in vivo from individual blood vessels. Enhancement of image signals and reduction of damage through adaptive shaping of excitation pulses will be investigated. PUBLIC HEALTH RELEVANCE: The availability of reliable, inexpensive, turn-key sources of high-energy ultrashort pulses will facilitate the development of biomedical imaging techniques with enhanced capabilities, and will enable the proliferation of nonlinear microscopies beyond research laboratories and into clinics. This will aid in the diagnosis and treatment of disease.